Three dimensional microtissue bioprinter

ABSTRACT

A bioprinter comprises one or more dispensing units. Each dispensing unit may include (i) a syringe including a hollow body and a plunger dimensioned to translate in the body wherein the body has an exit orifice; (ii) an actuator in contact with a proximal end of the plunger; (iii) a controller for moving the actuator; and (iv) a nozzle having a wall defining a fluid path extending from an inlet of the nozzle to an outlet of the nozzle. The inlet of the nozzle is in fluid communication with the exit orifice of the syringe body. The nozzle includes a fluid passageway in fluid communication with a source of fluid and the fluid path. The bioprinter can be used in a method of preparing microtissue comprising dispensing a bioink from one or more dispensing units of the bioprinter on a plate. The microtissue may comprise cartilage cells or tumor cells or liver cells.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/757,461 filed on Mar. 5, 2018, which is a U.S. NationalPhase of PCT Application No. PCT/US2016/050167 filed on Sep. 2, 2016,which claims priority from U.S. patent application Ser. No. 62/214,551filed on Sep. 4, 2015.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The invention relates to three-dimensional bioprinting, and moreparticularly to a computer controlled programmable three dimensional(3D) bioprinter generating live microtissues with precisely controlledmicroscale accurate XYZ motion and volumetric nanoliter dispensingcapability.

2. Description of the Related Art

Bioprinters were developed to try and meet the challenge of printingthree dimensional tissues. Bioprinters fabricate structures via adropwise printing of cells with a material which serves as the“bio-glue”. Bioprinters are limited by slow throughput inherent in thesmall size of their building materials as well as the vast number ofbuilding units that must be deposited. Various techniques andtechnologies exist for various applications of bioprinting.

Contact bioprinting is polymer printing without cells, and is used bythe 3D Bioplotter available from the Envisiontec company. The polymerprinting method only prints a scaffold with a plastics-like polymer.

Live cell bioprinting uses contact live cell bioprinting in bulk sizes.This technique does not allow microtissue printing as the resultanttissues are a couple of centimeters in size. Contact live cellbioprinting can only perform low throughput printing.

Non-contact valve based bioprinting techniques provide cell onlybioprinting. See, for example, Faulkner-Jones, et al., “Development of avalve-based cell printer for the formation of human embryonic stem cellspheroid aggregates”, Biofabrication 5.1 (2013): 015013. Non-contactvalve based bioprinting techniques can also provide cell and hydrogelbioprinting. See, for example, Moon, et al., “Layer by layerthree-dimensional tissue epitaxy by cell-laden hydrogel droplets”,Tissue Engineering Part C: Methods 16.1 (2009): 157-166. Moon, et al.printed the droplets into a larger piece and their system could onlygenerate a structure with cells suspended in the hydrogel without tissuearchitecture or morphology. Other commercial printers are unable toprint tissue with architecture and morphology similar to native tissues.

Air driven dispensing can provide coaxial gas flow bead generation. Thistechnique uses continuous air flow and continuous dispensing materialflow in a coaxial setup, which cannot control the volume of each dropletand cannot dispense a single droplet. This technique ordinarilydispenses thousands of droplets at a time, and in commercial systems,such as those available from Nisco Engineering AG, the material islimited to alginate.

In cell aggregates-non-real 3D models, cell aggregates are generated bya hanging-drop-method or from a non-adherent cell culture surface. Thistechnique has been used by others trying to mimic 3D tissue and thereare commercial products available. However, the aggregates are justpiled two dimensional cells without extracellular matrix. For example,it has been shown that the drug resistance mechanism of cell aggregatesis the same as two dimensional confluent cells. See, for example,Steadman, Kenneth, et al. “PolyHEMA spheroids are an inadequate modelfor the drug resistance of the intractable solid tumors.” Cell Cycle 7.6(2008): 818-829. Also, two dimensional cell culture does not resemblethe complex biological or pathological nature in vivo. Animal models donot fully resemble human diseases because of species differences.

From the clinical perspective, minimally invasive approaches are alwayspreferred for joint and many bone surgeries. Others have injectedchondrocytes taken from the knee and mesenchymal stem cells in the hopesof repairing tissue defects. However, weak mechanical properties, volumeshrinkage and long time in vivo growth make it inapplicable to jointcartilage or bone repair.

Therefore, there exists a need for a technology for printing humantissues in vitro that resemble the nature of human tissues in vivo.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a bioprinter comprisingone or more dispensing units. Each dispensing unit may include (i) asyringe including a hollow body and a plunger dimensioned to translatein the body wherein the body has an exit orifice; (ii) an actuator incontact with a proximal end of the plunger; (iii) a controller formoving the actuator; and (iv) a nozzle having a wall defining a fluidpath extending from an inlet of the nozzle to an outlet of the nozzle.The inlet of the nozzle is in fluid communication with the exit orificeof the body of the syringe. The nozzle includes a fluid passageway influid communication with a source of fluid and the fluid path. Thebioprinter can be used in a method of preparing microtissue comprisingdispensing a bioink from one or more dispensing units of the bioprinteron a plate. The microtissue may comprise stem cells, bone cells,cartilage cells, liver cells, tumor cells, tumor stromal cells,endothelial cells, and other types of cells. The bioprinter can be usedin a method for repairing a bone defect or a cartilage defect comprisingdispensing bioink from one or more dispensing units of the bioprinter tocreate microtissue comprising cartilage cells; and implanting themicrotissue in a bone defect or a cartilage defect. The bioprinter canbe used in a method for high-throughput screening for drug discoverycomprising dispensing bioink from one or more dispensing units of thebioprinter to create a plurality of microtissue samples; and contactingat least a portion of the plurality of microtissue samples with a drugcandidate compound. The microtissue samples may comprise tumor cells.

In this aspect, the controller may execute a program received from acomputer in the controller to drive the actuator toward the proximal endof the plunger to dispense a bioink from the body of the syringe. Thebioprinter may further comprise a temperature controller surrounding thebody of the syringe, a temperature controlled plate for receiving abioink from the outlet of the nozzle, and a humidifier for creating ahumidity controlled zone adjacent to the temperature controlled plate.In the bioprinter, fluid from the fluid passageway can separate dropletsof dispensed material and may force the droplets of dispensed materialto exit the outlet of the nozzle. In the bioprinter, a volume of eachdroplet may be in a range of 10 nanoliters to 15 microliters. The sourceof fluid may comprise a controllable value for supplying fluid. Thecontrollable value may be controlled by a second controller. The sourceof fluid in the bioprinter may comprise a controllable valve forsupplying pulsed fluid, pulsed air, millisecond pulsed air andsub-millisecond pulsed air. In the bioprinter, each dispensing unit maybe a non-contact dispensing unit, the nozzle may be integral with thesyringe body, and the nozzle may be separate from the syringe body. Theoutlet of the nozzle can have an inner diameter of 100 microns to 3millimeters. The bioprinter may comprise a plurality of dispensingunits, and each of the dispensing units may be mounted on an XYZ motionsystem.

In another aspect, the present disclosure provides a bioprintercomprising one or more dispensing units. Each dispensing unit includes(i) a syringe including a hollow body and a plunger dimensioned totranslate in the body, wherein the body has an exit orifice; (ii) anactuator in contact with and moving a proximal end of the plunger; (iii)a controller for controlling the motion of the actuator; and (iv) anozzle having a wall defining a fluid path extending from an inlet ofthe nozzle to an outlet of the nozzle. The inlet of the nozzle is influid communication with the exit orifice of the body of the syringe.The syringe does not include a valve between the exit orifice of thebody of the syringe and proximal end of the plunger. The bioprinter canbe used in a method of preparing microtissue comprising dispensing abioink from one or more dispensing units of the bioprinter on a plate.The microtissue may comprise stem cells, bone cells, cartilage cells,liver cells, tumor cells and other types of cells. The bioprinter can beused in a method for repairing a bone or cartilage defect comprisingdispensing bioink from one or more dispensing units of the bioprinter tocreate microtissue comprising cartilage cells; and implanting themicrotissues in a bone or cartilage defect. The bioprinter can be usedin a method for high-throughput screening for drug discovery comprisingdispensing bioink from one or more dispensing units of the bioprinter tocreate a plurality of microtissue samples; and contacting at least aportion of the plurality of microtissue samples with a drug library tofind candidate compounds. The microtissue samples may comprise tumorcells.

In this version of the bioprinter, the controller may execute a programstored in the controller to drive the actuator toward the proximal endof the plunger to dispense a bioink from the body of the syringe. Thebioprinter may further comprise a temperature controller surrounding thebody of the syringe, a temperature controlled plate for receiving abioink from the outlet of the nozzle, and a humidifier for creating ahumidity controlled zone adjacent to the temperature controlled plate.The nozzle in the bioprinter may include a fluid passageway in fluidcommunication with a source of fluid and the fluid path. The fluid fromthe fluid passageway may separate droplets of dispensed material and mayforce the droplets of dispensed material to exit the outlet of thenozzle. In the bioprinter, a volume of each droplet may be in a range of10 nanoliters to 15 microliters. The source of fluid may comprise acontrollable value for supplying fluid. The controllable value may becontrolled by a second controller. The source of fluid in the bioprintermay comprise a controllable valve for supplying pulsed fluid, pulsedair, millisecond pulsed air and sub-millisecond pulsed air. In thebioprinter, each dispensing unit may be a non-contact dispensing unit,the nozzle may be integral with the syringe body, and the nozzle may beseparate from the syringe body. The outlet of the nozzle can have aninner diameter of 100 microns to 3 millimeters. The bioprinter maycomprise a plurality of dispensing units, and each of the dispensingunits is mounted on an XYZ motion system.

One version of the bioprinter of the present disclosure is a computercontrolled programmable 3D bioprinter generating live micro tissues withprecisely controlled micro-scale accurate XYZ motion and volumetricnanoliter dispensing capability.

One version of the bioprinter of the present disclosure directly printshuman cells and extra cellular matrix mixtures onto the surface of cellculture containers, such as culture dishes and micro plates. The printedtissues have the morphology and function of native tissues. Thebioprinter works in two modes: (1) high speed printing mode which isused to bioprint micro tissues in large amount for the purpose of tissueregeneration; and (2) precision print mode, which bioprints one, or aspecific number of, microtissue(s) into each well of a 96 or 384 wellplate for drug screening and personalized therapeutic purposes.

Our bioprinter has a computer controlled XYZ linear motion system, whichcontrols the position and the movement of the aligned dispensing units(e.g., printing heads). Each dispensing unit dispenses our tissue ofinterest including cells or matrix or a specified mixture of the two(bio-ink) using the following mechanism. The dispensing unit is composedof: (1) a linear actuator-driven syringe pump controlled by a computer,(2) a dispensing syringe, which holds the bio-ink and dispenses apre-determined nanoliter-volume material through the nozzle each time;and (3) a novel dispensing nozzle. Because an adhesion force existsbetween the nozzle and the dispensed material, without external force,only larger droplets, about 50 microliters and above, can be generatedby gravity force alone. We use a millisecond pulse air dispensing unitto blow the nanoliter level dispensed material away from the syringenozzle and dispense it onto the surface. The short pulse air flow hasvery accurate positioning and does not interfere with the alreadydispensed droplets.

The printed tissues are in high throughput format for drug screening.The bioprinter rapidly prints live and functional micro tissues fortissue regeneration and modeling. The printed tissues can be composedof: (1) primary type of cells, such as tumor cells, hepatocytes andcardiomyocytes, (2) supporting cells, and (3) extracellular matrix sothat they function as native tissues because the micro environment ofthe tissues are reconstructed. Additionally, we can bioprint stem cellswith matrix for cartilage and bone regeneration. We theorize that we canbioprint fibroblasts capable of producing elastin, a molecule thatdiminishes through life resulting in wrinkles. This could have aprofound impact on cosmetic surgery. The ability to print a variety ofother tissues is limitless.

The volumetric non-contact dispensing unit is superior to existingdispensing technology. The volumetric dispensing unit extrudes cellsand/or matrix mixture out of the dispensing nozzle at nanoliterresolution; the extrusion is accomplished via a linear actuator. Becausethe extruded cell-matrix mixture cannot fall off the nozzle by gravitywhen the volume is at nanoliter level, our dispensing unit usesmillisecond air pulses to blow it away from the nozzle to accomplish thedispensing. Other non-contact printer heads, which are valve-based orpiezoelectric, rely on timing or pressure to control the volume ofdispensing. They need calibration for different viscosity materialswhile ours does not.

We are able to print micro tissues with morphology similar to the nativetissue. In order to accomplish this, we print the droplets of bioinkonto a temperature controlled surface to partially solidify the printedbio-ink during printing and control humidity to keep the printed bioinkfrom drying and to maintain high cell viability. Our precise technologyallows us to print tissue systems morphologically comparable to nativetissues.

Our system can use a coaxial setup or a non-coaxial setup for deliveryof pulsed air. A non-coaxial setup has better position control of thedispensed material.

Our results show that our printed tumor micro tissues, with the samecomponents as native tumor tissue, are much more drug resistant tochemotherapy drugs than the cell aggregates. Additionally, there aremany types of cells that do not aggregate and are not amenable toconventional cell aggregating techniques. We can still print thesenon-aggregating tissues using our bioprinter/technique. This representsa much more realistic testing scenario.

Our bioprinted microtissue is predeveloped and mechanically enhanced invitro. Once injected in vivo, the tissues self-assemble into largetissue amalgamations which can repair the defects.

Our bioprinted microtissue fills a need in predicting drug effectivenessor other medication on the human organism.

These and other features, aspects, and advantages of the presentinvention will become better understood upon consideration of thefollowing detailed description, drawings, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a bioprinter according to the inventionwith a partially exploded section showing the dispensing unit of thebioprinter.

FIG. 2 is a partial side view, with the dispensing unit incross-section, of the bioprinter of FIG. 1.

FIG. 2A is a perspective view of the XYZ motion system of the bioprinterof FIGS. 1 and 2.

FIG. 3 shows a schematic diagram of one version of a 3D bioprinter ofthe invention bioprinting mesenchymal stem cell microtissue, and thebioprinted mesenchymal stem cell microtissues.

FIG. 4 shows that mesenchymal stem cells (MSCs) in the microtissuesexhibit high cell viability after bioprinting (A) and injection througha 1.4 millimeter (mm) inside diameter (I.D.) needle (B). The scale baris 100 μm.

FIG. 5 shows confocal images (A 3D; B section) that revealed thehistological architecture of high cell density and extracellular matrix(ECM). The scale bar is 100 μm.

FIG. 6 shows that the dimension of MSC tissues stabilized after 3 daysof culture, which is the time point chosen for implantation.

FIG. 7 shows that MSC microtissues out grew into the surroundinghydrogel as shown by phase contrast imaging. The scale bar is 250 μm.

FIG. 8 shows MSCs that were labeled with Cellbrite red, green and bluefluorescent dyes and bioprinted into a individual microtissuerespectively. The tissue-fusion was observed when they were placedadjacently. The scale bar is 500 μm.

FIG. 9 shows that the printed microtissues could pass through a 1.4 mmI.D. transparent needle. The scale bar is 500 μm. MSCs in themicrotissues showed high cell viability after injection as shown in FIG.4B.

FIG. 10 shows that cartilage defects (2.0 mm, A-left shows the defect)were generated in the porcine articular cartilage explants. Stereo(A-right) and fluorescent microscopic (B) images of the defect show thefilling with MSC microtissues.

FIG. 11 shows that two weeks after implantation, the defect is stillcompletely filled in the microtissue filled group (A) comparing withlarge proportion of void space in the MSCs and hydrogel filled group (B)because of gel shrinkage. (C) is the non-treated group.

FIG. 12 shows that two weeks after implantation, the explants weresliced perpendicularly along the line as shown in (A). The slicedtissues were stained with cell viability dyes. B is a fluorescent imageof section interface (arrow pointing the bottom of the defect). Thegreen fluorescence indicates high cell viability.

FIG. 13 shows a schematic diagram of a 3D bioprinter of the invention.

FIG. 14 shows in (A), H&E staining of a bioprinted tumor microtissue(TMT), which has similar morphology to the native tumor tissues.(Bar=100 μm); in (B), 3D volume reconstructed image of a bioprinted TMT,which was imaged by confocal microscopy; and in (C), a representativeimage of a micro tumor tissue in a well of a 384 well plate, greenstaining indicates live cells. (Bar=100 μm)

FIG. 15 shows representative images of a TMT in (A), composed of humanendothelial cells in (B), human mesenchymal stem cells in (C) and humancancer cells in (D). (Bar=100 μm).

FIG. 16 shows an invasion assay. A TMT invaded into the surroundingmatrix after being cultured overnight. The red area (center) in C showsthe original location of the TMT at day 1. (Bar=100 μm)

FIG. 17 shows the TMT sections that were stained with aHypoxyprobe—Green kit which can stain a hypoxia area into a fluorescentgreen color. A large number of hypoxic cells were observed within thecore of the bioprinted micro tumor tissue. (Bar=100 μm)

FIG. 18 shows endothelial microtissues that were bioprinted and embeddedin the collagen matrix. When co-cultured with a bioprinted tumormicrotissue (arrow), a endothelial microtissue robust capillarysprouted. The TMT shows higher pro-angiogenic effect than endothelialmicrotissue alone or with vascular endothelial growth factorstimulation.

FIG. 19 shows a chemosensitivity analysis using two dimensional tumorcells (A) and TMT (B) in a 384-well plate. After 72 hours of drugtreatment, live/dead cell staining with Hoechst dye and propidium iodide(PI) were performed. With confocal or multiple-photon microscopy,optical sections were used for cell viability analysis and to determinethe IC₅₀. (Bar=50 μm)

FIG. 20 shows tumor micro tissue implantation that was performed using aCAM model. The stereo microscopic views at the times of (A) implantation(B) 8 days after implantation. In (C), the tumor tissue was surgicallyremoved with surrounding membrane. (Bar=500 μm)

FIG. 21 shows vascularized human micro liver tissues that werebioprinted with HepG2 human liver cells and human endothelial cells. Thevascularized micro liver tissues show significant higher cell density(A: H&E staining) and viability (B: live-dead staining, blue indicatinglive cells and purple indicating dead cells) comparing withnon-vascularized micro liver tissues (C : H&E staining. D: live-deadstaining).

Like reference numerals will be used to refer to like parts from Figureto Figure in the following description of the drawings.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to FIGS. 1 and 2, there is shown one non-limiting exampleembodiment of a bioprinter 10 according to the invention. The bioprinter10 may include a plurality of dispensing units 12. For ease ofillustration and description, one dispensing unit 12 is shown in FIG. 1.The dispensing unit 12 includes a syringe 14, a nozzle 25, an actuationmechanism 36, and a temperature controller 43 (shown in dashed lines inFIG. 1).

The syringe 14 has a body 15 defining a hollow interior space 16 of thesyringe 14. Typically, the body 15 comprises an opaque, translucent, ortransparent polymeric material, such as polypropylene. A plunger 17having a disk-shaped proximal end 18 and a disk-shaped distal end 19 ispositioned in the interior space 16 of the syringe 14 for movementtoward and away from an exit orifice 20 defined by a dispensing tip 21of the syringe 14. A bioink 22 is contained in the interior space 16 ofthe syringe 14. No valve is required between the exit orifice 20 of thebody 15 of the syringe 14 and the distal end 19 of the plunger.

The nozzle 25 has a side wall 26, an inlet 27, and an outlet 28, whichmay have an inside diameter of 100 microns to 3 millimeters. The sidewall 26 defines a fluid path 29 in the nozzle 25 between the inlet 27and the outlet 28. The nozzle 25 includes a fluid port 31 that defines afluid passageway 32 in fluid communication with the fluid path 29 in thenozzle 25. A fluid conduit 33 places the fluid passageway 32 of thefluid port 31 in fluid communication with a source of fluid 34. Acontrollable valve can be placed between the fluid conduit 33 and thesource of fluid 34 for supplying fluid to the fluid passageway 32 of thefluid port 31. The controllable valve can be controlled by a controllerthat opens and closes the controllable valve to deliver pulsed fluid(e.g., air) from the source of fluid 34 to the fluid passageway 32 ofthe fluid port 31. In the version of the dispensing unit 12 shown, thenozzle 25 is separate from the body 15 of the syringe 14. However, thenozzle 25 can be integral with the body 15 of the syringe 14.

The actuation mechanism 36 of the dispensing unit 12 includes acontroller 37 and an actuator 38 having a leadscrew 39 and a disk shapeddistal end section 41. The controller 37 may be linked to the actuator38 through an electrical cable 42. When the syringe 14 is installed inthe dispensing unit 12, the distal end section 41 of the actuator 38 isplaced in contact with the proximal end 18 of the plunger 17. Theactuator 38 is a linear actuator. The actuator 38 is a step motor baseddevice. Through the leadscrew 39 and rotating locker mechanism inside,the actuator 38 converts the rotation of a step motor inside theactuator 38 into the linear motion of the leadscrew 39. The distancewhich the distal end 41 travels depends on the degree of step motorrotation. The degree of rotation is controlled by the controller 37which receives the command sent from a computer program. The leadscrew39 moves toward the proximal end 18 of the plunger 17 to dispense thebioink 22 from the body 15 of the syringe 14.

The temperature controller 43 of the dispensing unit 12 includes ahousing 44 that surrounds temperature controlling elements 45, such asPeltier cooling-and-heating elements. The temperature controller 43provides a temperature controlled enclosure to surround the syringe 14and dispensing nozzle 25. One purpose of the temperature controlledenclosure is to control the temperature of the bioink 22 (e.g., cellsand/or matrix) at a desired level, which is cold most of time and may bewarmed occasionally according to different applications.

Still referring to FIGS. 1 and 2, the bioprinter 10 includes atemperature control plate 50 for receiving bioink 22 that is dispensedfrom the dispensing unit 12. The temperature control plate 50 maycomprise resistive heating elements or Peltier cooling-and-heatingelements. that receive electricity via an electrical lead 51. Thebioprinter 10 also includes a humidifier 58 with a tubular flow director59. The humidifier 58 creates a humidity controlled zone 61 adjacent thetemperature control plate 50. Droplets 66 of the bioink 22 exit outlet28 of the nozzle 25 and create microtissue 68 on the temperature controlplate 50.

Looking at FIG. 2A, the bioprinter 10 includes an XYZ motion system 80having a support X, a support Y₁, a support Y₂, and housings Z1, Z2, Z3,which each house a dispensing unit 12 as shown in FIG. 1. A drivecontrol mechanism 85 controls motion of support Y₁, and support Y₂towards and away from each other in directions Ay and By shown in FIG.2. A drive control mechanism 86 controls motion of support X indirections Ax and Bx shown in FIG. 2. A drive control mechanisms 87 a,87 b, 87 c control motion of housings Z1, Z2, Z3 in directions Az and Bzshown in FIG. 2. The drive control mechanisms 85, 86, 87 a, 87 b, 87 cmay be in electrical communication with a programmable controller forcontrolling XYZ motion of the housings Z1, Z2, Z3 (which each house adispensing unit 12). Each dispensing unit 12 is mounted to a Z-axis.

In FIG. 2A, three Z axis dispensing unit housings Z1, Z2, Z3 are shownas a non-limiting representative number. However, there is no limitationon the number of dispensing units; it depends on how many components areneeded to print for a specific microtissue. For example, when usingthree dispensing units, (i) three dispensing units could each includethe same bioink, (ii) two dispensing units could include the same bioinkand the other dispensing unit could include a different bioink, or (iii)three dispensing units could each include a different bioink.

Having described the components of the bioprinter 10, operation of thebioprinter 10 can be explained further. A microplate including an arrayof wells (e.g., 384, 1536, or 3456 wells) can be placed on thetemperature control plate 50 for receiving bioink 22 that is dispensedfrom the dispensing unit 12. Alternatively, other cell culturecontainers without an array of wells (such as a Petri dish or singlewell plate) on the temperature control plate 50 can directly receivebioink 22 that is dispensed from the dispensing unit 12.

Syringes 14 filled with the same or different bioinks are installed ineach dispensing unit 12, and the controller 37 is programmed for adispensing sequence. The controller 37 places the distal end section 41of each actuator 38 in contact with the proximal end 18 of theassociated plunger 17. The XYZ motion system 80 moves the dispensingunits over selected wells (or selected locations on the temperaturecontrol plate 50). As the controller drives each actuator 38 toward theplate 50, bioink 22 flows into the fluid path 29 in the nozzle 25. Thecontrollable valve then opens and closes to deliver pulsed fluid (e.g.,air) from the source of fluid 34 to the fluid passageway 32 of the fluidport 31. The pulsed fluid creates separate droplets 66 from the flow ofbioink in the fluid path 29 in the nozzle 25. A volume of each dropletcan be in a range of 10 nanoliters to 15 microliters. The droplets 66are repeatedly placed on the plate 50 (or in the wells of themicroplate) to create the microtissue samples 68 (see FIG. 2). Thebioprinter 10 uses volumetric based dispensing, which means the volumeof dispensing is linear to the distance that the plunger 17 moves.

The invention is further illustrated in the following Examples which arepresented for purposes of illustration and not of limitation.

EXAMPLES Example 1

Example 1 describes the development of injectable mesenchymal stem cells(MSCs) microtissues for repairing cartilage defects using 3Dbioprinting. This is a representative project for our high speedbioprinting in which we bioprint micro-cartilage and bone tissues usinghuman bone marrow mesenchymal stem cells (MSCs). We have successfullybioprinted micro bone tissue and MSC-incorporated-tissue for cartilageand bone regeneration. Our bioprinted micro-tissue is predeveloped andmechanically enhanced in vitro. Once injected in vivo, the tissuesself-assemble into large tissue amalgamations which can repair thedefects.

Introduction

Articular cartilage exhibits poor intrinsic capacity for repair, andtissue engineering is a new approach for articular cartilage repair.Bone marrow mesenchymal stem cells (MSCs) and bulk hydrogel have beenused to repair the cartilage defects in open surgery. As a minimallyinvasive approach is always preferred for joint surgery, injectabletissue engineered cartilage would be an ideal solution to repaircartilage defects. However, volume shrinkage and poor mechanicalproperties of hydrogel, which results in void space in the implantation,limit the application of a MSCs-hydrogel injection. In this example, weaimed to develop volume stabilized MSC microtissues using 3Dbioprinting, and repair the defect in articular cartilage through theself-assembly of the micro tissues.

Methods

1. 3D bioprinting: The microtissues, comprising human MSCs and bioinkcomposed of hydrogels in the pre-optimized combination, were directlybioprinted using our custom developed 3D bioprinter of the presentinvention (see FIG. 3).

2. Cell viability: Live/dead cell viability assay in MSC microtissueswas performed after bioprinting.

3. Morphology: Morphology of the microtissue was studied by HE stainingand confocal microscopy.

4. Dimension alteration: Diameters of the microtissues were imaged bymicroscopically at a series of time points to analyze the volumealteration.

5. Single tissue 3D out growth: The microtissues were embedded in thehydrogel within a 96-well plate to observe tissue outgrowth under aninverted microscope.

6. Multiple tissue fusion/assembly: MSCs were labeled with Cellbrite(red, green and blue respectively) and several microtissues were placedon the ultra-low attachment surface to observe tissue fusion atdifferent time points.

7. Injectability: The bioprinted microtissues suspended in medium werepushed through a 1.4 mm I.D. needle, and cell viability was analyzed bylive/dead staining.

8. Cartilage defect repairing in a cartilage explants culture model:Defects, 2 mm in diameter, were made in the porcine articular cartilageexplants. The defects were filled with MSC microtissues to evaluateintegration outcome. MSC hydrogel were used as the control. Also,live/dead staining was performed to study cell viability after 2 weekscultivation.

Results

FIG. 3 shows a schematic diagram of one version of a 3D bioprinter ofthe invention bioprinting mesenchymal stem cell microtissue, and thebioprinted mesenchymal stem cell microtissues.

FIG. 4 shows that mesenchymal stem cells (MSCs) in the microtissuesexhibit high cell viability after bioprinting (A) and injection througha 1.4 millimeter (mm) inside diameter (I.D.) needle (B). The scale baris 100 μm.

FIG. 5 shows confocal images (A 3D; B section) that revealed thehistological architecture of high cell density and ECM. The scale bar is100 μm.

FIG. 6 shows that the dimension of MSC tissues stabilized after 3 daysof culture, which is the time point chosen for implantation.

FIG. 7 shows that an MSC microtissue out grew into the surroundinghydrogel as shown by phase contrast imaging. The scale bar is 250 μm.

FIG. 8 shows MSCs that were labeled with Cellbrite red, green and bluefluorescent dyes and bioprinted into a individual microtissuerespectively. The tissue-fusion was observed when they were placedadjacently. The scale bar is 500 μm.

FIG. 9 shows that the printed microtissues could pass through a 1.4 mmI.D. transparent needle. The scale bar is 500 μm. MSCs in themicrotissues showed high cell viability after injection as shown in FIG.4B.

FIG. 10 shows that cartilage defects (2.0 mm, A-left shows the defect)were generated in the porcine articular cartilage explants. Stereo(A-right) and fluorescent microscopic (B) images of the defect show thefilling with MSC microtissues.

FIG. 11 shows that two weeks after implantation, the defect is stillcompletely filled in the microtissue filled group (A) comparing withlarge proportion of void space in the MSCs and hydrogel filled group (B)because of gel shrinkage. (C) is the non-treated group.

FIG. 12 shows that two weeks after implantation, the explants weresliced perpendicularly along the line as shown in (A). The slicedtissues were stained with cell viability dyes. B is a fluorescent imageof section interface (arrow pointing the bottom of the defect). Thegreen fluorescence indicates high cell viability.

Conclusion

In Example 1, we have successfully developed a new approach in 3Dbioprinting to generate MSC microtissues and it shows promise to repairthe articular cartilage defects minimally invasively.

Example 2

Example 2 describes high throughput 3D bioprinting of tumor microtissues for drug development and personalized cancer therapy.

Introduction

Cancer caused about 25% of all deaths in the United States in 2014. Invitro anti-cancer drug screening is widely used in the pharmaceuticalindustry and chemosensitivity test for patients. However, only 2D assaysand simple 3D culture models were used previously. But these models donot resemble the native tumor microenvironment, or yield inaccurateprediction of drug sensitivity. In this example, we report a novel 3Dbioprinted model of tumor microtissues (TMTs), which resemble the nativetumor characteristics, for high throughput screening. We characterizedthe 3D bioprinted tumors in various of aspects.

Methods

3D bioprinting: The bioink composed of tumor cells, stromal cells andECM is loaded into our custom developed 3D bioprinter of the presentinvention. Micro tumor tissues were directly bioprinted into each wellof a 96 or 384 well plate. The viability of the cells in the tissueswere analyzed using live/dead staining. FIG. 13 shows the schematicdiagram of the 3D bioprinter.

Morphologic characterization: The TMTs were cryosectioned or opticalsectioned, and analyzed by hematoxylin and eosin (H&E or HE) staining,confocal and multiple-photon microscopy respectively.

Hypoxic micro environment analysis: TMT cryosections were stained byusing Hypoxyprobe-Green kit which labels hypoxia area with greenfluorescence color.

Invasion assay: The TMTs were first printed and the extra bioink withoutcells were printed around the tumor tissues. Phase contrast orfluorescent imaging were used to analyze tumor invasion into the matrix.

Angiogenesis assay: In order to analyze the pro-angiogenic biomoleculessecreted by tumor tissues. Bioprinted microtissue consisted only HumanUmbilical Vein Endothelial Cells (HUVECs) and matrix were imbedded incollagen hydrogel and co-cultured with bioprinted TMT and theangiogenesis sprouting was analyzed by phase contrast and fluorescencemicroscopy.

Chemosensitivity test: To determine the chemosensitivity of 3Dbioprinted TMT and conventional 2D cells, cells and TMTs were platedinto each well of a 384-well plate. Drugs were added for 72 hours andlive/dead fluorescent was performed. Confocal or multiple-photonmicroscopy were used to analyze the cell viability in 3D and 2Drespectively.

In vivo implantation: Chorioallantoic membrane assay (CAM) was used toevaluate the tumorigenicity in vivo. TMTs were implanted on the top ofthe growing CAMs on 8th day of development. On the 16th day, each eggwas imaged using stereo microscope. The tumors were surgically removedwith surrounding membrane and fixed in 4% paraformaldehyde for furtherhistological analysis.

Results

FIG. 14 shows in (A), H&E staining of a bioprinted tumor microtissue(TMT), which has similar morphology to the native tumor tissues.(Bar=100 μm); in (B), 3D volume reconstructed image of a bioprinted TMT,which was imaged by confocal microscopy; and in (C), a representativeimage of a micro tumor tissue in a well of a 384 well plate, greenstaining indicates live cells. (Bar=100 μm)

FIG. 15 shows representative images of a TMT in (A), composed of humanendothelial cells in (B), human mesenchymal stem cells in (C) and humancancer cells in (D). (Bar=100 μm).

FIG. 16 shows an invasion assay. A TMT invaded into the surroundingmatrix after being cultured overnight. The red area (center) in C showsthe original location of the TMT at day 1. (Bar=100 μm)

FIG. 17 shows the TMT sections that were stained with aHypoxyprobe—Green kit which can stain a hypoxia area into a fluorescentgreen color. A large number of hypoxic cells were observed within thecore of the bioprinted micro tumor tissue. (Bar=100 μm)

FIG. 18 shows endothelial microtissues that were bioprinted and embeddedin the collagen matrix. When co-cultured with a bioprinted tumormicrotissue (arrow), an endothelial microtissue robust capillarysprouted. The TMT shows higher pro-angiogenic effect than endothelialmicrotissue alone or with vascular endothelial growth factorstimulation.

FIG. 19 shows a chemosensitivity analysis using two dimensional tumorcells (A) and TMT (B) in a 384-well plate. After 72 hours of drugtreatment, live/dead cell staining with Hoechst dye and propidium iodide(PI) were performed. With confocal or multiple-photon microscopy,optical sections were used for cell viability analysis and to determinethe IC₅₀. (Bar=50 μm)

FIG. 20 shows tumor micro tissue implantation that was performed using aCAM model. The stereo microscopic views at the times of (A) implantation(B) 8 days after implantation. In (C), the tumor tissue was surgicallyremoved with surrounding membrane. (Bar=500 μm)

Conclusions

We have successfully developed a high throughput 3D bioprinting systemto generate tumor micro tissues in vitro, which resemble manycharacteristics of the native tumor tissues. The system has thereforedemonstrated as a powerful tool for screening anti-cancer drugs andselecting the sensitive drugs for personalized cancer therapy.

Example 3

We 3D bioprinted liver tissues. Specifically, we have successfullybioprinted vascularized human micro liver tissues using HepG2 humanliver cells and human endothelial cells. As shown in FIG. 21, thevascularized micro liver tissues show significant higher cell density(A) and viability (B) comparing with non- vascularized micro livertissues (C and D).

Thus, the invention provides a three dimensional microtissue bioprinterand methods for using the three dimensional microtissue bioprinter. Wehave disclosed a computer controlled programmable 3D bioprinter forgenerating live micro tissues with precisely controlled micro-scaleaccurate XYZ motion and volumetric nanoliter dispensing capability. Wehave printed many types of tissue with many different types of cellsincluding pluripotent stem cells, bone marrow mesechymal stem cells,osteoblasts, fibroblast, human endothelial cells, liver cells, and manydifferent tumor cells.

We have bioprinted micro tumor tissues for high throughput drugscreening and therapeutic purposes. We have characterized many aspectsof the printed tumor tissues including morphology, function, biomarker,micro-environment, 3D structure, and invasion and drug sensitivities. Aswe have experience in tumor bioprinting, we envision successful microheart and liver tissue printing which requires the same technicalprocedures with different cells. We 3D bioprinted liver tissues.

We have performed anti-tumor drug screening with 3D bioprinted tumortissues as described above. This is a desirable service for thepharmaceutical industry to develop new anti-cancer drugs, including butnot limited to, chemotherapy drugs, and radiotherapy sensitivityenhancing drugs. The advantage is that our system can assist indevelopment significantly more quickly and at markedly lower developmentcosts (no animal studies). The 3D nature of the bioprinted tissuesshould also allow for more accurate assessment compared to currentlyemployed 2D cultured cell screening also saving money and time.

The invention could be used for personalized cancer therapy. Threedimensional bioprinting tumor tissues using cancer cells isolated fromindividual patients after surgery or biopsy can be used to determinesensitive drug(s) or drug combination(s) for the individual patient,including but not limited to, chemotherapy drugs andradiotherapy-enhancing drugs. Because the 3D tumor tissue closelyresembles the in vivo situation, it can more accurately help theoncologists to find the best medication regimen for the patient.

The invention could be used for cartilage and bone tissue regenerationthrough minimally invasive surgery.

The invention could be used for drug metabolism testing for thepharmaceutical industry. We can utilize our 3D bioprinted micro livertissues to test drugs under development for their metabolism profiles inhigh throughput.

The invention could be used for drug toxicity screening for thepharmaceutical industry. We can utilize our 3D bioprinted micro liverand heart tissues to screen for toxicity of candidate drugs for thepharmaceutical industry.

Although the present invention has been described in detail withreference to certain embodiments, one skilled in the art will appreciatethat the present invention can be practiced by other than the describedembodiments, which have been presented for purposes of illustration andnot of limitation. Therefore, the scope of the appended claims shouldnot be limited to the embodiments contained herein.

What is claimed is:
 1. A method of preparing microtissue, the methodcomprising: (a) providing a bioprinter comprising: one or moredispensing units, each dispensing unit including: (i) a syringeincluding a hollow body and a plunger dimensioned to translate in thebody, the body having an exit orifice; (ii) an actuator in contact witha proximal end of the plunger; (iii) a controller for controlling linearmotion of the actuator; and (iv) a nozzle having a wall defining a fluidpath extending from an inlet of the nozzle to an outlet of the nozzle,the inlet of the nozzle being in fluid communication with the exitorifice of the body of the syringe, wherein the nozzle includes a fluidpassageway in fluid communication with a source of fluid and the fluidpath; and (b) dispensing a bioink from one or more dispensing units ofthe bioprinter on a plate.
 2. The method of claim 1 wherein: themicrotissue comprises cartilage cells.
 3. The method of claim 1 wherein:the microtissue comprises tumor cells, tumor stromal cells andendothelial cells.
 4. The method of claim 1 wherein: the microtissuecomprises liver cells.
 5. A method for repairing a bone defect or acartilage defect, the method comprising: (a) providing a bioprintercomprising: one or more dispensing units, each dispensing unitincluding: (i) a syringe including a hollow body and a plungerdimensioned to translate in the body, the body having an exit orifice;(ii) an actuator in contact with a proximal end of the plunger; (iii) acontroller for controlling linear motion of the actuator; and (iv) anozzle having a wall defining a fluid path extending from an inlet ofthe nozzle to an outlet of the nozzle, the inlet of the nozzle being influid communication with the exit orifice of the body of the syringe,wherein the nozzle includes a fluid passageway in fluid communicationwith a source of fluid and the fluid path; (b) dispensing bioink fromone or more dispensing units of the bioprinter to create microtissuecomprising cartilage cells; and (c) implanting the microtissue in thebone defect or the cartilage defect.
 6. A method of high-throughputscreening for drug discovery, the method comprising: (a) providing abioprinter comprising: one or more dispensing units, each dispensingunit including: (i) a syringe including a hollow body and a plungerdimensioned to translate in the body, the body having an exit orifice;(ii) an actuator in contact with a proximal end of the plunger; (iii) acontroller for controlling linear motion of the actuator; and (iv) anozzle having a wall defining a fluid path extending from an inlet ofthe nozzle to an outlet of the nozzle, the inlet of the nozzle being influid communication with the exit orifice of the body of the syringe,wherein the nozzle includes a fluid passageway in fluid communicationwith a source of fluid and the fluid path; (b) dispensing bioink fromone or more dispensing units of the bioprinter to create a plurality ofmicrotissue samples; and (c) contacting at least a portion of theplurality of microtissue samples with a drug candidate compound.
 7. Themethod of claim 6 wherein: the microtissue samples comprise tumor cells.8. A bioprinter comprising: one or more dispensing units, eachdispensing unit including: a syringe including a hollow body and aplunger dimensioned to translate in the body, the body having an exitorifice; (ii) an actuator in contact with a proximal end of the plunger;(iii) a controller for moving the actuator; and (iv) a nozzle having awall defining a fluid path extending from an inlet of the nozzle to anoutlet of the nozzle, the inlet of the nozzle being in fluidcommunication with the exit orifice of the body of the syringe, whereinthe syringe does not include a valve between the exit orifice of thebody of the syringe and a distal end of the plunger.
 9. The bioprinterof claim 8 wherein: the controller executes a program stored in thecontroller to drive the actuator toward the proximal end of the plungerto dispense a bioink from the body of the syringe.
 10. The bioprinter ofclaim 8 further comprising: a temperature controller surrounding thebody of the syringe.
 11. The bioprinter of claim 8 further comprising: atemperature controlled plate for receiving a bioink from the outlet ofthe nozzle.
 12. The bioprinter of claim 11 further comprising: ahumidifier for creating a humidity controlled zone adjacent to thetemperature controlled plate.
 13. The bioprinter of claim 8 wherein: thenozzle includes a fluid passageway in fluid communication with a sourceof fluid and the fluid path, and fluid from the fluid passagewayseparates droplets of dispensed material and forces the droplets ofdispensed material to exit the outlet of the nozzle.
 14. The bioprinterof claim 13 wherein: a volume of each droplet is in a range of 10nanoliters to 15 microliters.
 15. The bioprinter of claim 13 wherein:the source of fluid comprises a controllable valve for supplying fluid.16. The bioprinter of claim 15 wherein: the controllable valve iscontrolled by a second controller.
 17. The bioprinter of claim 13wherein: the source of fluid comprises a controllable valve forsupplying pulsed fluid.
 18. The bioprinter of claim 13 wherein: thesource of fluid comprises a controllable valve for supplying pulsed air.19. The bioprinter of claim 13 wherein: the source of fluid comprises acontrollable valve for supplying millisecond pulsed air.
 20. Thebioprinter of claim 13 wherein: each dispensing unit is a non-contactdispensing unit.
 21. The bioprinter of claim 8 wherein: the nozzle isintegral with the syringe body.
 22. The bioprinter of claim 8 wherein:the nozzle is separate from the syringe body.
 23. The bioprinter ofclaim 8 wherein: the outlet of the nozzle has an inner diameter of 100microns to 3 millimeters.
 24. The bioprinter of claim 8 wherein: thebioprinter comprises a plurality of the dispensing units.
 25. Thebioprinter of claim 8 further comprising: each of the dispensing unitsis mounted on an XYZ motion system.
 26. A method of preparingmicrotissue, the method comprising: (a) dispensing a bioink from one ormore dispensing units of the bioprinter of claim 8 on a plate.
 27. Themethod of claim 26 wherein: the microtissue comprises cartilage cells.28. The method of claim 26 wherein: the microtissue comprises tumorcells.
 29. The method of claim 26 wherein: the microtissue comprisesliver cells.
 30. A method for repairing a bone defect or a cartilagedefect, the method comprising: (a) dispensing bioink from one or moredispensing units of the bioprinter of claim 8 to create microtissuecomprising cartilage cells; and (b) implanting the microtissue in thebone defect or the cartilage defect.
 31. A method of high-throughputscreening for drug discovery, the method comprising: (a) dispensingbioink from one or more dispensing units of the bioprinter of claim 8 tocreate a plurality of microtissue samples; and (b) contacting at least aportion of the plurality of microtissue samples with a drug candidatecompound.
 32. The method of claim 31 wherein: the microtissue samplescomprise tumor cells.